SOX-10 and TRP-1 expression in feline ocular and nonocular melanomas.

In felines, ocular and nonocular melanomas are uncommon tumors that represent a diagnostic challenge for pathologists, especially when amelanotic. To date, the immunohistochemical diagnostic panel in cats is based on specific melanocytic markers (Melan-A and PNL2) and a nonspecific but sensitive marker (S100). In human medicine, SOX-10 is reported to be a sensitive antibody for the detection of melanoma micrometastasis in the lymph node. TRP-1, an enzyme involved in melanogenesis, has recently been used in humans and dogs as a specific melanocyte marker. The aim of this study was to evaluate the cross-reactivity and the expression of SOX-10 and TRP-1 antibodies in feline normal tissue and melanocytic tumors. Thirty-one cases of ocular, cutaneous, and oral melanomas were retrospectively evaluated and confirmed by histopathological examination and by immunolabeling with Melan-A and/or PNL2. SOX-10 nuclear expression in normal tissues was localized in epidermal, subepidermal, hair bulb, and iridal stromal melanocytes and dermal nerves. In melanomas, nuclear expression of SOX-10 was detected in ocular (11/12; 92%), oral (6/7; 86%), and cutaneous sites (12/12; 100%). TRP-1 cytoplasmic immunolabeling in normal tissue was observed in epidermal and bulbar melanocytes and in the lining pigmented epithelium of the iris and in its stroma. Its expression was positively correlated to the degree of pigmentation in the tumor and was observed in 75% of ocular (9/12), 43% of oral (3/7), and 33% of cutaneous melanomas (4/12). This study demonstrated the cross-reactivity of SOX-10 and TRP-1 antibodies in feline non-neoplastic melanocytes and their expression in ocular and nonocular melanomas.

Vulvo-vaginal epithelial tumors in mares: A preliminary investigation on epithelial-mesenchymal transition and tumor-immune microenvironment.

Vulvo-vaginal epithelial tumors are uncommon in mares, and data on the epithelial-to-mesenchymal transition (EMT) and the tumor-immune microenvironment (TIME) are still lacking. This is a study investigating the equus caballus papillomavirus type 2 (EcPV2) infection state as well as the EMT process and the tumor microenvironment in vulvo-vaginal preneoplastic/ benign (8/22) or malignant (14/22) epithelial lesions in mares. To do this, histopathological, immunohistochemical, transcriptomic, in situ hybridization, and correlation analyses were carried out. Immunohistochemistry quantification showed that cytoplasmic E-cadherin and ß-catenin expression as well as nuclear ß-catenin expression were features of malignant lesions, while benign/preneoplastic lesions were mainly characterized by membranous E-cadherin and ß-catenin expression. Despite this, there were no differences between benign and malignant equine vulvo-vaginal lesions in the expression of downstream genes involved in the canonical and noncanonical wnt/ß-catenin pathways. In addition, malignant lesions were characterized by a lower number of cells with cytoplasmic cytokeratin expression as well as a slightly higher cytoplasmic vimentin immunolabeling. The TIME of malignant lesions was characterized by more numerous CD204+ M2-polarized macrophages. Altogether, our results support the hypothesis that some actors in TIME such as CD204+ M2-polarized macrophages may favor the EMT process in equine vulvo-vaginal malignant lesions providing new insights for future investigations in the field of equine EcPV2-induced genital neoplastic lesions.

Genetic Characterization of a Novel Equus caballus Papillomavirus Isolated from a Thoroughbred Mare.

Papillomaviruses (PVs) are small, non-enveloped viruses, ubiquitous across the animal kingdom. PVs induce diverse forms of infection, such as cutaneous papillomas, genital papillomatosis, and carcinomas. During a survey on the fertility status of a mare, a novel Equus caballus PV (EcPV) has been identified using Next Generation Sequencing, and it was further confirmed with genome-walking PCR and Sanger sequencing. The complete circular genome 7607 bp long shares 67% average percentage of identity with EcPV9, EcPV2, EcPV1, and EcPV6, justifying a new classification as Equus caballus PV 10 (EcPV10). All EcPV genes are conserved in EcPV10, and phylogenetic analysis indicates that EcPV10 is closely related to EcPV9 and EcPV2, genus Dyoiota 1. A preliminary EcPV10 genoprevalence study, carried out on 216 horses using Real Time PCRs, suggested a low incidence of this isolate (3.7%) compared to EcPVs of the same genus such as EcPV2 and EcPV9 in the same horse population. We hypothesize a transmission mechanism different from the one observed in the closely related EcPV9 and EcPV2 that particularly infect Thoroughbreds. This horse breed is usually submitted to natural mating, thus indicating a possible sexual diffusion. No differences were detected for breeds in terms of susceptibility to EcPV10. Further studies are needed to investigate the molecular mechanisms behind the host and EcPV10 infection to explain the reduced viral spread.

Canine melanocytes: Immunohistochemical expression of melanocytic markers in different somatic areas.

BACKGROUND: Melanoblasts originate in the neural crest from where they migrate to peripheral tissues and differentiate into melanocytes. Alteration during melanocyte development and life can cause different diseases, ranging from pigmentary disorders and decreased visual and auditory functions, to tumours such as melanoma. Location and phenotypical features of melanocytes have been characterised in different species, yet data on dogs are lacking. OBJECTIVE: This study investigates the expression of melanocytic markers Melan A, PNL2, TRP1, TRP2, SOX-10 and MITF in melanocytes of selected cutaneous and mucosal surfaces of dogs. ANIMALS: At necropsy, samples from five dogs were harvested from oral mucosa, mucocutaneous junction, eyelid, nose and haired skin (abdomen, back, pinna, head). MATERIALS AND METHODS: Immunohistochemical and immunofluorescence analyses were performed to assess marker expression. RESULTS: Results showed variable expression of melanocytic markers in different anatomical sites, particularly within epidermis of haired skin and dermal melanocytes. Melan A and SOX-10 were the most specific and sensitive melanocytic markers. PNL2 was less sensitive, while TRP1 and TRP2 were seldomly expressed by intraepidermal melanocytes in haired skin. MITF had a good sensitivity, yet the expression often was weak. CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate a variable expression of melanocytic markers in different sites, suggesting the presence of subpopulations of melanocytes. These preliminary results pave the way to understanding the pathogenetic mechanisms involved in degenerative melanocytic disorders and melanoma. Furthermore, the possible different expression of melanocyte markers in different anatomical sites could influence their sensitivity and specificity when used for diagnostic purposes.

Tumor-Associated Macrophages in Canine Oral and Cutaneous Melanomas and Melanocytomas: Phenotypic and Prognostic Assessment.

The tumor microenvironment is a complex system, where neoplastic cells interact with immune and stromal cells. Tumor-associated macrophages (TAMs) are considered among the most numerically and biologically noteworthy cellular components in tumors and the attention on this cellular population has been growing during the last decade, both for its prognostic role and as a potential future therapeutic target. Melanoma, particularly the oral form, despite being one of the most immunogenic tumors, bears a poor prognosis in dogs and humans, due to its highly aggressive biological behavior and limited therapeutic options. The aims of this study are to characterize and quantify TAMs (using CD163, CD204, Iba1, and MAC387) in canine melanocytic tumors and to evaluate the association of these markers with diagnosis, histologic prognostic features, presence of metastases, and outcome, and to provide preliminary data for possible future therapies targeting TAMs. Seventy-two melanocytic tumors (27 oral melanomas, 25 cutaneous melanomas, 14 cutaneous melanocytomas, and 6 oral melanocytomas) were retrospectively selected and submitted to immunohistochemistry and double immunofluorescence. Double immunolabeling revealed that most CD163+ and CD204+cells co-expressed Iba1, which labeled also dendritic cells. Iba1 was instead rarely co-expressed with MAC387. Nevertheless, the expression of macrophagic markers showed a mild to moderate association among the four markers, except for CD204 and MAC387. The number of CD163+, CD204+, and MAC387+ cells was significantly higher in oral melanomas compared to oral melanocytomas (p < 0.001; p < 0.05 and p < 0.01, respectively), whereas Iba1 was differentially expressed in cutaneous melanomas and melanocytomas (p < 0.05). Moreover, CD163, IBA1 and MAC387 expression was associated with nuclear atypia and mitotic count. The number of CD163+cells was associated with the presence of metastases and tumor-related death in oral melanocytic tumors (p < 0.05 and p = 0.001, respectively).

Detection of Equus Caballus Papillomavirus Type-2 in Asymptomatic Italian Horses.

Equine Papillomavirus 2 (EcPV2) is responsible for squamous cell carcinomas (eSCCs) of external genitalia of both male and female horses. However, few studies report the EcPV2 prevalence among healthy horses. Currently, the lack of these data does not permit identifying at-risk populations and, thus, developing screening protocols aimed at the early detection of the infection, as for humans. The aim of our study was to estimate the genoprevalence of EcPV2 in clinically healthy horses in Italy and to evaluate their innate immune response. For this purpose, penile and vulvar swabs of 234 healthy horses were collected through sampling with sterile cytobrushes. Nucleic acids were isolated and EcPV2-L1 presence (DNA) and gene expression (RNA) were checked by RT-qPCR. Our results showed EcPV2-L1 DNA presence in 30.3% of the samples and L1 expression in 48% of the positive samples. No statistically significant differences were found in genoprevalence in relation to sex, age, and origin, while, concerning breeds, the Thoroughbred had the highest risk of infection. Concerning specifically the mares, 40.2% of them resulted in being positive for EcPV2; our findings show a major positivity in pluriparous (p = 0.0111) and mares subjected to natural reproduction (p = 0.0037). Moreover, samples expressing L1 showed an increased expression of IL1B (p = 0.0139) and IL12p40 (p = 0.0133) and a decreased expression of RANKL (p = 0.0229) and TGFB (p = 0.0177). This finding suggests the presence of an effective immune response, which could explain the low incidence of SCCs in positive horses, despite a high EcPV2 genoprevalence (30%).

A case of generalised cutaneous apocrine cystomatosis in a Pekingese dog.

Clinical, histological and immunohistochemical examination of a 13-year-old male client-owned Pekingese dog revealed an uncommon presentation of apocrine cutaneous cystomatosis. This is a rare non-neoplastic condition of uncertain cause, characterised by multiple cystically dilated apocrine sweat glands. We aimed to describe the features of this unusual case of generalised cutaneous apocrine cystomatosis in the dog, which can be useful to distinguish it from multifocal benign cystic apocrine tumours.

Non Epitheliotropic B-Cell Lymphoma with Plasmablastic Differentiation vs. Cutaneous Plasmacytosis in a 12-Years-Old Beagle: Case Presentation and Clinical Review.

Cutaneous lymphoid neoplasms and cutaneous plasmacytosis are rare in the dog; in human and in veterinary medicine, these have many clinical, cytological, histological, and phenotypic similarities, and a diagnosis of certainty is not easy. The aim of this study is to describe a case of cutaneous non epitheliotropic B-cell lymphoma (CNEBL) with plasmablastic differentiation vs. multiple cutaneous plasmacytosis (CP) in a dog, since the scarce bibliographic data on these topics. A 12-year-old male Beagle dog was presented for multiple, nodular, cutaneous, and subcutaneous, indolent masses disseminated on the whole body. Cytological, histological, flow cytometric, and immunohistochemical examinations, as well as complete radiographic evaluation, echocardiography, and abdominal ultrasound were performed. Cytology, histopathology, flow cytometric, and immunohistochemical examination, performed on the skin lesions, revealed a B-cell phenotype with plasmablastic differentiation. Nevertheless, a final diagnosis could not be achieved and it was categorized as a case of borderline CNEBL with plasmablastic differentiation versus CP. The dog was treated with a COP chemotherapeutic protocol. Total remission was obtained and relapse occurred 120 days later. To our knowledge, specific markers are actually unavailable to certainly differentiate CNEBL and CP in the dog and future studies are needed to improve knowledge on these pathologies in veterinary medicine, since prognosis and therapy are different.

Tumor-infiltrating lymphocytes (TILs) in feline melanocytic tumors: A preliminary investigation.

The presence and the role of tumor-infiltrating lymphocytes (TILs) in different types of tumors, but particularly in melanoma, has become more and more investigated during the last decade, both in human and veterinary medicine. Melanocytic tumors are quite rare in cats, with diffuse iris melanoma being the most commonly diagnosed in this species. The aim of this study was to characterize the lymphocytic infiltration in feline melanocytic tumors and to analyze their association with the histological features of malignancy recognized in these tumors, as well as with the expression of the most commonly used immunohistochemical markers. Thirty-eight feline melanocytic tumors were retrospectively selected; histological and immunohistochemical characterization of the tumors (histologic criteria of malignancy; S100, Melan A, and PNL2 expression) and of TILs (presence/absence, density, distribution, and grade; CD3, CD20 expression) were performed and associations between them tested. Results showed that TILs grade increased with cellular pleomorphism (P < 0.05) and, within the group of cutaneous melanocytic tumors, also with the mitotic count (P < 0.05). On the other hand, TILs grade was inversely associated with the percentage of neoplastic cells positive for Melan A (P < 0.05) and PNL2 (P < 0.05). Both CD3+ and CD20+ lymphocytes increased significantly with TILs grade and in association with mitotic count, when stratified in low/high quantity. This preliminary study suggests that TILs in feline melanoma may be associated with histologic features of malignancy and loss of melanocytic-specific markers, such as Melan A and PNL2. Further studies, with a larger cohort and follow-up information, are recommended.

Investigation of the Epithelial to Mesenchymal Transition (EMT) Process in Equine Papillomavirus-2 (EcPV-2)-Positive Penile Squamous Cell Carcinomas.

Equine penile squamous cell carcinoma (epSCC) is the most frequent tumor of the external male genitalia, representing 67.5% of equine genital cancers. epSCC is associated with papilloma virus (PV) infection and has been recently proposed as a model for human PV-induced squamous cell carcinomas. It has already been suggested that epSCC might undergo epithelial-to-mesenchymal transition (EMT). This work aims to investigate in detail this process and the possible role of PV oncoproteins in epSCC. For this purpose, 18 penile SCCs were retrospectively selected and tested for both EcPV2 presence and oncoproteins (EcPV2 E6 and EcPV2 E7) expression. Moreover, immunohistochemical EMT characterization was carried out by analyzing the main epithelial markers (E-cadherin, ß-catenin, and pan-cytokeratin AE3/AE1), the main mesenchymal markers (N-cadherin and vimentin), and the main EMT-related transcription factors (TWIST-1, ZEB-1). PCR analysis was positive for EcPV2 in 16 out of 18 samples. EMT was investigated in epSCC positive for EcPV2. The immunohistochemistry results suggested the presence of EMT processes in the neoplastic cells at the tumor invasive front. Moreover, the significant upregulation of RANKL, together with BCATN1, LEF1, and FOSL1 genes, might suggest a canonical Wnt pathway activation, similarly to what is reported in human penile squamous cell carcinomas.

PD-L1/PD-1 and CTLA-4 Expression in Equine Penile Squamous Cell Carcinomas.

In horses, penile squamous cell carcinomas (epSCCs) are among the most common cutaneous neoplastic lesions. These tumors usually arise in benign lesions such as viral plaques and papillomas frequently induced by Equus caballus papillomavirus type 2 (EcPV2) infection. In the last decade, the introduction of immune checkpoint inhibitors (ICI) for the treatment of human cancers has demonstrated promising results. Among the most commonly targeted pathways, there is PD-1/PD-L1 and CTLA-4. The aim of this study is to investigate the expression of the PD-1/PD-L1 pathway and CTLA-4 in the tumor microenvironment of epSCCs to assess the feasibility of an immunotherapeutic approach. Twenty equine epithelial tumors were retrospectively selected and submitted to RT-qPCR for PD-1 and PD-L1 genes. After testing antibodies cross-reactivity by western blotting, immunohistochemistry for PD-L1 and CTLA-4 was performed. Results from RT-qPCR demonstrated that 3/20 cases expressed the PD-L1 gene, whereas the PD-1 gene was not detected. Immunohistochemical positivity for PD-L1 was found only in one case. CTLA-4-positive cells were observe in all cases but were few (Mdn = 4.8; IQR = 2.3-7.1 cells/HPF). In this study group, PD-1/PD-L1 and CTLA-4 do not appear to be highly expressed and therefore the use of ICI in epSCCs may not have promising rates of response.

A First Case Report of Orbital Extra-Adrenal Paraganglioma in Cat.

Paraganglioma is a rare neuroendocrine neoplasm originating from paraganglia and consisting of neuroendocrine cells of the sympathetic and parasympathetic nervous system. Extra-adrenal paraganglioma occurs with a low incidence in both humans and animals. This report presents the first case of paraganglioma in a cat with orbital primary location. An 18-year-old spayed female European domestic shorthair cat of 3.60 kg body weight was evaluated in a private veterinary clinic in Perugia, Italy, for a pronounced exophthalmos of the right eye. The cat underwent surgery for the enucleation of the right eye and of the mass. The biopsy samples of the removed tissue were fixed in 10% buffered neutral formalin for histological and immunohistochemical evaluations. Therefore, specific markers were used for immunohistochemical investigations, such as anti-neuron specific enolase (NSE), anti-synaptophysin, anti-glial fibrillary acid protein, anti-cytokeratin and anti-chromogranin. The results of these investigations allowed establishing the final diagnosis of ocular extra-adrenal paraganglioma of the cat.

Equine Genital Squamous Cell Carcinoma Associated with EcPV2 Infection: RANKL Pathway Correlated to Inflammation and Wnt Signaling Activation.

Equine genital squamous cell carcinomas (egSCCs) are among the most common equine tumors after sarcoids, severely impairing animal health and welfare. Equus caballus papillomavirus type 2 (EcPV2) infection is often related to these tumors. The aim of this study was to clarify the molecular mechanisms behind egSCCs associated with EcPV2 infection, investigating receptor activator of nuclear factor-kappa B ligand (RANKL) signaling in NF-kB pathway, together with the Wnt and IL17 signaling pathways. We analyzed the innate immune response through gene expression evaluation of key cytokines and transcription factors. Moreover, Ki67 index was assessed with immunohistochemistry. EcPV2-E6 DNA was checked, and viral presence was confirmed in 21 positive out to 23 cases (91%). Oncogene expression was confirmed in 14 cases (60.8%) for E6 and in 8 (34.7%) for E2. RANKL, nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB)-p50, NFKBp65, interleukin (IL)-6, IL17, IL23p19, IL8, IL12p35, IL12p40, ß-catenin (BCATN1), FOS like 1 (FOSL1), and lymphoid enhancer binding factor 1 (LEF1) showed a significant upregulation in tumor samples compared to healthy tissues. Our results describe an inflammatory environment characterized by the activation of RANKL/RANK and IL17 with the relative downstream pathways, and a positive modulation of inflammatory cytokines genes such as IL6 and IL8. Moreover, the increase of BCATN1, FOSL1, and LEF1 gene expression suggests an activation of both canonical and non-canonical Wnt signaling pathway that could be critical for carcinogenesis and tumor progression.

Characterization of Primary Cultures of Normal and Neoplastic Canine Melanocytes.

Although numerous animal models, especially mouse models, have been established for the study of melanoma, they often fail to accurately describe the mechanisms of human disease because of their anatomic, physiological, and immune differences. The dog, as a spontaneous model of melanoma, is nowadays considered one of the most valid alternatives due to the heterogeneity of clinical presentations and of histological and genetic similarities of canine melanoma with the human counterpart. The aim of the study was to optimize a protocol for the isolation and cultivation of healthy and neoplastic canine melanocytes derived from the same animal and obtained from cutaneous and mucosal (oral) sites. We obtained five primary tumor cell cultures (from 2 cutaneous melanoma, 2 mucosal melanoma and 1 lymph node metastasis) and primary normal melanocyte cell cultures (from normal skin and mucosa) from the same dogs. Immunocytochemical characterization with Melan A, PNL2 and S100 antibodies confirmed the melanocytic origin of the cells. This work contributes to expanding the case record of studies on canine melanoma cell cultures as suitable model to study human and canine melanoma. To the authors' knowledge, this is the first report of isolation of normal skin and mucosal canine melanocytes.

FoxP3, CTLA-4, and IDO in Canine Melanocytic Tumors.

Despite promising immunotherapy strategies in human melanoma, there are few studies on the immune environment of canine melanocytic tumors. In humans, the activation of immunosuppressive cell subpopulations, such as regulatory T cells (Tregs) that express forkhead box protein P3 (FoxP3), the engagement of immunosuppressive surface receptors like cytotoxic T lymphocyte antigen (CTLA-4), and the secretion of molecules inhibiting lymphocyte activation, such as indoleamine-pyrrole 2,3-dioxygenase (IDO), are recognized as immunoescape mechanisms that allow tumor growth and progression. The aim of our study was to investigate the expression of these immunosuppression markers in canine melanocytic tumors and to postulate their possible role in melanoma biology and progression. Fifty-five formalin-fixed, paraffin-embedded canine melanocytic tumors (25 oral melanomas; 20 cutaneous melanomas; 10 cutaneous melanocytomas) were selected to investigate the expression of FoxP3, CTLA-4, and IDO by immunohistochemistry and RT-qPCR (real-time quantitative polymerase chain reaction). All of the tested markers showed high gene and protein expression in oral melanomas and were differently expressed in cutaneous melanomas when compared to their benign counterpart. IDO expression was associated with an increased hazard of death both in univariable and multivariable analyses (P < .05). FoxP3 protein expression >6.9 cells/HPF (high-power field) was an independent predictor of death (P < .05). CTLA-4 gene and protein expressions were associated with a worse prognosis, but only in the univariable analysis (P < .05). FoxP3, CTLA-4, and IDO likely play a role in canine melanoma immunoescape. Their expression, if supported by future studies, could represent a prognostic tool in canine melanoma and pave the way to future immunotherapeutic approaches in dogs.

A five-year cohort study on testicular tumors from a population-based canine cancer registry in central Italy (Umbria).

Canine testicular tumors account for about 90 % of tumors affecting the male genitalia. Seminomas (SEM), Sertoli cell tumors (SCT), and interstitial cell tumors (ICT) are the most common histological diagnoses, but their incidence shows high variability among studies. Our aim is to report the results on the analysis of testicular tumors recorded by the population-based Umbria Canine Cancer Registry (CCR) for a 5-year time period and to assess the value of tumor major diameter, measured during trimming, in discriminating neoplastic from non-neoplastic lesions. The study was conducted on 388 testicular tumors (on 1969 total male tumors) diagnosed in 355 dogs from 2014 to 2018. The median incidence was 35 cases/100,000 dogs, with a proportion equal to 19,7 % of all tumors. The incidence on neutered male dogs was 352/100,000. Most tumors were ICTs (50 %), with fewer SEMs and SCTs (29 % and 17 %, respectively). Only 3 % of tumors were mixed germ cell-sex cord-stromal tumors (MGC-SCST). Ten percent of cases had multiple tumors in the same testicle, with SEM-ICT being prevalent (69.2 %). Tumors in cryptorchid testes were 5.9 % of the total, mostly SCT (60.9 %). Mean age at diagnosis was 10.7 ± 2.7, with similar values for different tumor types. Significant incidence ratios (IRR) were found in Golden retriever (IRR 7.18, CI95 % 4.72-10.92) and in English cocker spaniel (IRR 2.30, CI95 % 1.28-4.13) when compared with mixed breed dogs. A value of 0.3 cm (major diameter) of lesions at the moment of trimming was helpful in discriminating a final tumor histological diagnosis from a non-tumor lesion. Since the number of tumors included in this evaluation was limited, further studies to confirm the diagnostic value of this measure are recommended. Our results provided information on the incidence of canine testicular tumors in Umbria region that can be compared with future results and incidence from other geographical areas if provided with reliable data on the total population, can offer solid information on the incidence and proportion of different tumor types in specific territories, contributing also to the supervision of its inhabitants' health. Moreover, pathological data such as the major diameter of tumors can be obtained and contribute to diagnostic routine and standardization.

Equine Penile Squamous Cell Carcinomas as a Model for Human Disease: A Preliminary Investigation on Tumor Immune Microenvironment.

Penile squamous cell carcinomas (SCCs) are common tumors in older horses, with poor prognosis mostly due to local invasion and recurrence. These tumors are thought to be mainly caused by Equus caballus papillomavirus type 2 (EcPV-2). The aim of this study is to characterize the tumor immune environment (TIME) in equine penile tumors. Equine penile epithelial tumors (17 epSCCs; 2 carcinomas in situ, CIS; 1 papilloma, P) were retrospectively selected; immune infiltrate was assessed by histology and immunohistochemistry; RT-qPCR tested the expression of selected chemokines and EcPV-2 DNA and RNA. The results confirmed EcPV-2-L1 DNA in 18/20 (90%) samples. L1 expression was instead retrieved in 13/20 cases (65%). The samples showed an increased infiltration of CD3+lymphocytes, macrophages (MAC387; IBA1), plasma cells (MUM1), and FoxP3+lymphocytes in the intra/peritumoral stroma when compared to extratumoral tissues (p < 0.05). Only MAC387+neutrophils were increased in EcPV-2high viral load samples (p < 0.05). IL12/p35 was differentially expressed in EcPVhigh and EcPVlow groups (p = 0.007). A significant decrease of IFNG and IL2 expression was highlighted in TGFB1-positive samples (p < 0.05). IBA1 and CD20 were intratumorally increased in cases where IL-10 was expressed (p < 0.005). EpSCCs may represent a good spontaneous model for the human counterpart. Further prospective studies are needed in order to confirm these preliminary results.

E-Cadherin Expression in Canine Melanocytic Tumors: Histological, Immunohistochemical, and Survival Analysis.

E-cadherin, a glycoprotein involved in cell-cell adhesion, has a pivotal role in epithelial-mesenchymal transition, a process through which neoplastic epithelial cells develop an invasive phenotype. In human cutaneous melanomas, decreased E-cadherin expression is associated with shorter survival and increased Breslow thickness, whereas in the dog its role is poorly understood. Tumor thickness and modified Clark level were recently proposed as useful features to assess canine melanocytic tumors, but no studies investigated their association with E-cadherin expression. We performed immunohistochemistry on 77 formalin-fixed, paraffin-embedded primary canine melanocytic tumors. A 3-tier and a 2-tier classification system for assessing E-cadherin expression were tested, with the latter being more informative for the assessment of canine melanocytic tumors. E-cadherin expression was lower in cutaneous melanomas than melanocytomas, as well as in amelanotic tumors compared to pigmented tumors. In amelanotic melanomas, absent E-cadherin expression was associated with an unfavorable outcome, suggesting a potential use of this marker in defining the prognosis of amelanotic melanomas. E-cadherin expression was lower in tumors with greater tumor thickness and modified Clark level ≥IV, suggesting its possible utility in identifying the most invasive tumors. The expression of E-cadherin in oral melanomas was heterogeneous, but was associated with pigmentation and clinical outcome; thus, E-cadherin evaluation could be advantageous to detect the most aggressive neoplasms. However, cutaneous melanomas without E-cadherin expression frequently had a favorable clinical outcome. Hence, its importance as prognostic factor should be carefully considered depending on the tumor origin.

Equus caballus papillomavirus type 2 (EcPV2) in co-occurring vulvar and gastric lesions of a pony.

In horses, squamous cell carcinomas (SCCs) are the most common malignant tumors developing on non-pigmented skin, muco-cutaneous areas, like external genitalia, and, less frequently, in the stomach. Growing evidence suggests Equus caballus papillomavirus type 2 (EcPV2) as causative agent of genital SCCs. Our case report describes a 20-year-old, female, mixed-breed pony with co-occurring vulvar papilloma and in situ carcinoma and gastric SCC. Both lesions were positive for the same EcPV2, as confirmed by DNA sequencing. E6 mRNA expression was observed both in vulvar lesions and gastric SCC, while L1 mRNA was expressed in the vulvar tumor. To the best of the Authors' knowledge, this is the first report of an association between EcPV2 and equine gastric squamous cell carcinoma, with co-occurring EcPV2-positive genital lesions. Further studies are required to assess the real prevalence and the possible role of this viral type in these equine tumors.

Cystic thymic degeneration with pseudoepitheliomatous hyperplasia in a Beagle puppy: an idiopathic lesion?

A 6-mo-old female Beagle dog was inappetent and depressed. The radiographic, ultrasonographic, and computed tomographic examination of the chest revealed a 10 × 7 cm multicystic mediastinal structure interpreted as altered thymus, in association with moderate pleural effusion that laboratory tests confirmed as hemothorax. No history of trauma or anticoagulant drug intoxication was reported, and no coagulation disorders were detected. Afterward, medial cranial sternotomy was performed to remove the altered tissue. Histologically, this tissue was compatible with a thymic remnant, characterized by numerous cystic lesions, mostly blood filled and lined by flattened-to-cuboidal epithelial cells, occasionally projecting into the surrounding stroma, and forming cytokeratin-positive ribbons, trabeculae, and papillae. Lymphocytes were scant, and numerous areas of congestion and hemorrhage were present throughout the samples. This case of idiopathic thymic hemorrhage with cystic degeneration of the thymus and pseudoepitheliomatous hyperplasia was an incidental finding; the dog recovered from surgery uneventfully.